DNA polymerases provide a canon of strategies for translesion synthesis past oxidatively generated lesions

Abstract

Deducing the structure of the DNA double helix in 1953 implied the mode of its replication: Watson-Crick (WC) base pairing might instruct an enzyme, now known as the DNA polymerase, during the synthesis of a daughter stand complementary to a single strand of the parental double helix. What has become increasingly clear in the last 60 years, however, is that adducted and oxidatively generated DNA bases are ubiquitous in physiological DNA, and all organisms conserve multiple DNA polymerases specialized for DNA synthesis opposite these damaged templates. Here, we review recent crystal structures depicting replicative and bypass DNA polymerases encountering two typical lesions arising from the oxidation of DNA: abasic sites, which block the replication fork, and the miscoding premutagenic lesion 7,8-dihydro-8-oxoguanine (8-oxoG).

Figure 1

(A) Premutagenic DNA lesions form at guanine nucleotides by several mechanisms. Spontaneous depurination constitutes one such mechanism giving rise to abasic sites. 8-oxoG, the product of oxidation of the guanine nucleobase, becomes protonated at the N7. (B) When paired with dAMP, replicative polymerases translocate the 8-oxoG mismatch without triggering 3’→5’ exonuclease activity. The extraneous O8 mimics the N3 of an undamaged purine and is therefore able to interact appropriately with conserved H-bond donors in the palm domain. (C) Y-family polymerases encode an additional domain, the PAD, which facilitates TLS. This example depicts Dpo4 stabilizing the anti form of 8-oxoG by juxtaposing an arginine residue between the 5’ phosphate and the O8 of 8-oxoG such that the lesion pairs with dCMP.

Figure 2

Replicative and specialized polymerases process abasic sites (indicated by black arrows) differently by partitioning the template strand into alternative conformations. Rev1 employs H-bonds (dashed lines) from R357 to direct incorporation of dCTP, while extruding the furan template (PDBID 3OSP [19]). Polymerase β requires R283 to stack under the abasic site for efficient selection of dATP and alignment of the primer terminus of this gapped duplex DNA substrate (PDBID 3ISD [21]). A single hydrogen bond is also observed form R283 to the phosphate backbone. Polymerase II flips out three nucleotides, including the abasic site, and utilizes an upstream (+3) nucleotide for incorporation of dATP in this particular example (PDBID 3K5L [28]). Dpo4, like pol II, flips out the abasic site and allows an upstream (+1) guanine to pair with dCTP in the polymerase active site (PDBID 1S0N [27]). Polymerase ι squeezes the active site such that the incoming nucleotide stacks with the template strand (−1 base) and engages in van der Waals contacts with the lesion itself (PDBID 3G6X [33]). Pol ι inserts dGTP slightly more readily than other nucleotides due the H-bond depicted between Q59 and the exocyclic N2. The replicative polymerase RB69 gp43 does not utilize any of these template rearrangements. 5-NITP, a non natural dATP analog, protrudes its nitro moiety into the void created by the presence of the abasic site (PDBID 2OZM [15]). This substituent stacks with the template (−1 base), which is reminiscent of nucleotide incorporation opposite an abasic site by pol ι.

Figure 3

Klentaq stalls while inserting dATP opposite an abasic site. (A) The binary complex illustrates the conformation of helix O and Y671 while the fingers are fully open (PDBID 4KTQ [56]). (B) In the stalled complex where furan occupies the preinsertion site, a hydrogen bond is formed between Y671 and dATP (PDBID 3LWL [24]). At this point, approximately 5.9Å separate the α-phosphate and the C3’ at the primer terminus, making nucleophilic attack unlikely in this intermediate conformation. (C) In the closed ternary complex, the distance between the α-phosphate and the primer terminus is 3.6 Å, showing that full closure of the fingers domain reduces this distance by ~2.3 Å from the stalled complex (PDBID 1QSY [25]). (D, insert) The superposition of binary, furan, and ternary structures shows that the furan complex represents a stalled complex. The closure of the fingers domain provides a twisting motion at Y671 that moves this conserved residue away from the insertion site such that the template can interrogate the nucleotide in the closed complex. Binary and furan complexes were superimposed onto the ternary complex, achieving an RMSD on protein atoms in core regions of 0.97 and 0.68 Å, respectively.

Figure 4

Dpo4 contributes R332 to stabilize 8-oxoG in the anti conformation, such that the Watson-Crick face of the lesion is available for hydrogen bonding with dCMP (PDBID 2XCP [57]). Although the PAD of polymerase η does not contact the nucleotide in the insertion site, pol η makes two contacts from asparagine residues to the O8 of 8-oxoG in the translocated complex (PDBID 3OHB [50]). This stabilization is likely important for extension of the error-free incorporation product. Polymerase κ accommodates 8-oxoG in its active site, except the syn form is preferred (PDBID 3IN5 [53]). This establishes Hoogsteen base pairing with dATP, which can lead to G→T transversion mutations. Polymerase ι achieves error-free insertion opposite 8-oxoG (syn) by a unique mode of Hoogsteen base pairing with dCTP. Polymerase ι has a propensity to bind purines in the syn configuration in its very narrow active site cleft (PDBID 3Q8P [55]).